The efficient folding of large, multi-domain proteins in the cytosol is accomplished by chaperones. Hsp90 is a chaperone that confers in vivo activity upon a select set of client proteins. Hsp90 folds signal transduction molecules, tRNA synthetases, and oncoproteins. Hsp90 folds these proteins in a process driven by ATP hydrolysis and facilitated by co-chaperones. Upon ATP binding, hsp90 undergoes a large conformational change where one set of chaperones is exchanged for another. Hsp90 inhibition by ATP analogs blocks tumor growth with a significant therapeutic index. Three structural aspects of hsp90 function are intriguing. What are the structural changes induced in the client protein during hsp90 folding? How does hsp90 recognize a diverse set of proteins and distinguish between single mutant forms of others? What conformation is assumed by hsp90 bound by ATP analogs? To determine the structural dynamics ofhsp90 and its clients we will employ MPAX, a new technique that measures changes in protein structure, flexibility, and solvent accessibility. An MPAX experiment tetermines the extent that protein structure protects individual residues from solvent, analogous to the results of hydrogen-deuterium exchange experiments. We will use MPAX to determine the conformation of a bound hsp90 client, how hsp90 recognizes client proteins, and the conformation of inhibited hsp90.